T Cell Immunity in Connective Tissue Disease Patients Targets the RNA Binding Domain of the U1-70kDa Small Nuclear Ribonucleoprotein

Abstract

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.

U1 RNA induces innate immunity signaling

“…Using saturation binding studies, we previously demonstrated that use of our in vitro U1 RNA synthesis method yields a product with high affinity for U1-70 kd (8). For the current study, the in vitro synthesis method yielded a homogeneous product at high concentrations by agarose gel/ ethidium bromide visualization (results not shown), eliminating the possibility that contaminating RNA activity could be responsible for the stimulatory activities observed in our studies.…”

“…To create complexes of U1 RNA and 70-kd protein, 70 kd was produced and purified as a maltose binding protein fusion protein from Escherichia coli, as previously described (7). To create complexes, U1 RNA was incubated at room temperature for 20 minutes with an excess of 70 kd in PBS, as previously described (8).…”

“…Cells were incubated with U1 RNA or control stimuli in complete medium for 16 hours and pulsed with 3 H-labeled thymidine for the last 8 hours of culture. Cells were harvested, and incorporation of 3 H-labeled thymidine was measured by liquid scintillation counting, as previously described (8).…”

Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K

“…Importantly, the apoptosis-associated change in phosphorylation we identified involves the RRM of the U1-70K protein, which is the main target of SLE autoantibodies and autoreactive T cells. 16,17,20,21 In addition, we have previously reported the importance of the phosphorylated Ser140 residue in modulating the autoimmune response. 18,19 Therefore, we propose that the Materials and Methods Antibodies.…”

“…14,15 The U1-70K protein, in particular the RNA recognition motif (RRM), has been identified as an important target for both autoantibodies and CD4 þ autoreactive T cells from mice and patients with lupus. 16,17 We previously found that a peptide encompassing residues 131-151 in the RRM of the U1-70K protein and phosphorylated at Ser140 (peptide P140) was recognized by CD4 þ T cells from mice and patients with lupus and protected young lupus mice against the disease in contrast to the nonphosphorylated peptide, which was not tolerogenic. 18,19 In addition, for unknown reasons, the apoptotic form of the protein is a preferred target of autoantibodies.…”